Retroviral integrase (IN) catalyzes the essential step of integrating the double-stranded DNA copy of the viral genome into the chromosome of an infected cell. Certain mutations of HIV-1 IN can specifically impair reverse transcription with no apparent effects on other steps in the life cycle. However, the underlying mechanism for this defect is poorly understood. In vitro, HIV-1 IN physically interacts with reverse transcriptase (RT) and stimulates reverse transcription, but the biological relevance of this RT-IN interaction is not known. Using nuclear magnetic resonance spectroscopy, we have identified the RT-interacting surface on IN, and have determined the affinity and kinetics of RT-IN complex formation by using a surface plasmon resonance-based biosensor. In addition to RT interaction, we recently have characterized HIV-1 IN mutations that resulted in poor core yield and instability, and reduced incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to capsid (CA). Further analyses indicate that IN is required during uncoating by maintaining CypA-CA interaction, which promotes optimal stability of the viral core. Taken together, we hypothesize that HIV-1 IN can affect reverse transcription either directly by interacting with RT or indirectly by altering uncoating. The goal of this application is to gain a better understanding of the functional role of IN during uncoating and reverse transcription. The specific aims are (1) to determine the biological significance of the HIV-1 RT-IN interaction, (2) to characterize the physical interaction between HIV-1 RT and IN, and examine other factors that can modulate the RT-IN interaction, and (3) to understand the mechanism by which IN affects the uncoating of viral cores. In Aim 1, we will test the biological relevance of the RT-IN interaction during infection by disrupting the putative IN-RT binding interface and assessing the impact on reverse transcription and viral replication. We will also screen for RT mutants that can compensate for the RT- noninteracting IN mutations, and use a fluorescence-based high-throughput screen to identify small-molecule inhibitors of the RT-IN interaction and determine whether such inhibitors also block viral replication. In Aim 2, we will map the IN-interacting domain of RT using a targeted protein footprinting method. We will also examine the effect of a host factor SIP1, which binds specifically to IN and is required for reverse transcription, on the RT-IN binding interface, and determine if SIP1 can affect IN's activities on nuclear import and integration. In Aim 3, we will determine if the IN-CA interaction in the viral core requires post-translational modification or a bridge factor. We will also examine the effect of CypA- and TRIM51-binding to CA on IN's role during uncoating, and the relationship between uncoating and reverse transcription. In the process, we will shed light on the interactions among key retroviral proteins and their host factors, and the effect of such interactions on reverse transcription and uncoating. Characterization of the interactions and determination of their biological significance may reveal new functional roles for IN, and identify new potential targets for anti-HIV therapy.